Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus

Macdonald, Darel; Thomson, Darren D; Johns, Anna; Valenzuela, Adriana Contreras; Gilsenan, Jane Mabey; Lord, Kathryn M.; Bowyer, Paul; Denning, David W.; Read, Nick D.; Bromley, Michael

doi:10.1128/aac.01615-18

Cited by 22 publications

(29 citation statements)

References 53 publications

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“…An indication of one of the additional functions of NsdC came recently from a study aiming to identify new transcription factors involved in the calcium stress response in A. fumigatus (30). Large-scale phenotypic screenings have been widely used to identify new components of signaling pathways and to study drug resistance mechanisms (46)(47)(48)(49). A library of null-mutant transcription factors was challenged with high concentrations of CaCl 2 , and this resulted in the identification of an A. fumigatus DnsdC mutant with increased sensitivity to CaCl 2 relative to the wild-type parental strain, suggesting a role for NsdC in calcium response.…”

Section: Discussionmentioning

confidence: 99%

Novel Biological Functions of the NsdC Transcription Factor in Aspergillus fumigatus

Castro

Valero

Chiaratto

et al. 2021

mBio

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The fungal zinc finger transcription factor NsdC is named after, and is best known for, its essential role in sexual reproduction (never in sexual development). In previous studies with Aspergillus nidulans, it was also shown to have roles in promotion of vegetative growth and suppression of asexual conidiation. In this study, the function of the nsdC homologue in the opportunistic human pathogen A. fumigatus was investigated. NsdC was again found to be essential for sexual development, with deletion of the nsdC gene in both MAT1-1 and MAT1-2 mating partners of a cross leading to complete loss of fertility. However, a functional copy of nsdC in one mating partner was sufficient to allow sexual reproduction. Deletion of nsdC also led to decreased vegetative growth and allowed conidiation in liquid cultures, again consistent with previous findings. However, NsdC in A. fumigatus was shown to have additional biological functions including response to calcium stress, correct organization of cell wall structure, and response to the cell wall stressors. Furthermore, virulence and host immune recognition were affected. Gene expression studies involving chromatin immunoprecipitation (ChIP) of RNA polymerase II (PolII) coupled to next-generation sequencing (Seq) revealed that deletion of nsdC resulted in changes in expression of over 620 genes under basal growth conditions. This demonstrated that this transcription factor mediates the activity of a wide variety of signaling and metabolic pathways and indicates that despite the naming of the gene, the promotion of sexual reproduction is just one among multiple roles of NsdC. IMPORTANCE Aspergillus fumigatus is an opportunistic human fungal pathogen and the main causal agent of invasive aspergillosis, a life-threatening infection especially in immunocompromised patients. A. fumigatus can undergo both asexual and sexual reproductive cycles, and the regulation of both cycles involves several genes and pathways. Here, we have characterized one of these genetic determinants, the NsdC transcription factor, which was initially identified in a screen of transcription factor null mutants showing sensitivity when exposed to high concentrations of calcium. In addition to its known essential roles in sexual reproduction and control of growth rate and asexual reproduction, we have shown in the present study that A. fumigatus NsdC transcription factor has additional previously unrecognized biological functions including calcium tolerance, cell wall stress response, and correct cell wall organization and functions in virulence and host immune recognition. Our results indicate that NsdC can play novel additional biological functions not directly related to its role played during sexual and asexual processes.

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Section: Discussionmentioning

confidence: 99%

Novel Biological Functions of the NsdC Transcription Factor in Aspergillus fumigatus

Castro

Valero

Chiaratto

et al. 2021

mBio

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“…Aspergillus fumigatus is a filamentous fungus that causes severe allergy and life-threating infections, especially in susceptible and immunocompromised patients (Macdonald et al, 2019). The morbidity and mortality in such patients remain high (Brown et al, 2012), and therefore this mold is extensively studied in laboratories all over the world.…”

Section: Introductionmentioning

confidence: 99%

Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction

et al. 2019

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Aspergillus fumigatus is an opportunistic human pathogenic mold. DNA extraction from this fungus is usually performed by mechanical perturbation of cells, as it possesses a rigid and complex cell wall. While this is not problematic for single isolates, it can be time consuming for large numbers of strains if using traditional DNA extraction procedures. Therefore, in this article we describe a fast and efficient thermal‐shock method to release DNA from spores of A. fumigatus and other filamentous fungi without the need for complex extraction methods. This is especially important for high‐throughput PCR analyses of mutants in 96‐ or 384‐well formats in a very short period of time without any concern about sample cross‐contamination. This method is currently being used to validate the protein‐coding gene and non‐coding RNA knockout libraries in A. fumigatus generated in our laboratory, and could be used in the future for diagnostics purposes. © 2019 The Authors.

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“…The observed high frequency of diploids in CF patients may be due to local stress factors that induce and/or select for diploidy in A. fumigatus crucial for parasexual recombination ( figure 5 ). Indeed, nitrogen starvation, sublethal concentrations of the antifungal compound itraconazole or the inhibitory drug cerulenin were found to induce cell fusion and heterokaryon formation in isogenic A. fumigatus isolates in vitro [ 33 ]. In the absence of asexual and sexual sporulation as modes of generating genetic variation, the parasexual processes in long-lasting pulmonary mycelial biofilms can be highly relevant for gradual adaptation of A. fumigatus to the human host [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

Parasexual recombination enablesAspergillus fumigatusto persist in cystic fibrosis

et al. 2020

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Aspergillus fumigatus is a saprobic fungus that causes a range of pulmonary diseases, some of which are characterised by fungal persistence such as is observed in cystic fibrosis (CF) patients. Creation of genetic variation is critical for A. fumigatus to adapt to the lung environment, but biofilm formation, especially in CF patients, may preclude mutational supply in A. fumigatus due to its confinement to the hyphal morphotype. We tested our hypothesis that genetic variation is created through parasexual recombination in chronic biofilms by phenotypic and genetic analysis of A. fumigatus isolates cultured from different origins.As diploids are the hallmark of parasex, we screened 799 A. fumigatus isolates obtained from patients with CF, chronic pulmonary lung disease, acute invasive aspergillosis and from the environment for spore size. Benomyl sensitivity, nuclear content measurements through fluorescence activated cell sorting and scanning electron microscopy were used to confirm the diploid state of large size spores. Whole genome sequencing was used to characterise diploid associated genetic variation.We identified 11 diploids in isolates recovered from six of 11 (55%) CF-patients and from one of 24 (4%) chronic aspergillosis patients, but not in 368 isolates from acute infection and the environment. Diploid formation was associated with accumulation of mutations and variable haploid offspring including a voriconazole-resistant isolate.Parasexual recombination allows A. fumigatus to adapt and persist in CF patients, and plays a role in azole resistance development. Our findings are highly significant for understanding the genetics and biology of A. fumigatus in the human lung.

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Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus

Cited by 22 publications

References 53 publications

Novel Biological Functions of the NsdC Transcription Factor in Aspergillus fumigatus

Novel Biological Functions of the NsdC Transcription Factor in Aspergillus fumigatus

Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction

Parasexual recombination enablesAspergillus fumigatusto persist in cystic fibrosis

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